It has become apparent that the total cysteine content in plasma (tCcys) is an important parameter in evaluating the risk of cardiovascular disease. It has been reported that low risk of cardiovascular disease is associated with tCys levels at 250-275 μM whereas a higher risk is associated both with lower levels (<225 μM) and higher levels (>300 μM). El-Khairy, et al., Circulation (2001) 2544-2549. The levels of the cysteine homolog, homocysteine (tHcy) are also relevant as the risk of venous thrombosis and myocardial infarction are increased at high levels of homocysteine. Marcucci, et al., M. J. Clin. Pathol. (2001) 116:56-60. It appears that the levels of cysteine and homocysteine are interrelated since cysteine is, along with albumin, a covalent carrier of the homocysteine in circulation and cysteine is also a competitor of homocysteine for cellular uptake as well as a homocysteine metabolite via the transsulfuration pathway. Hortin, et al., J. Clin. Chem. (2001) 47:1121-1124.
It is also known that individuals with very high levels of homocysteine due to defects in the transsulfuration pathway have low levels of total cysteine. It is estimated that one percent of the population is defective in the enzyme responsible; homozygous deficiency results in homocysteinuria. The ratio of tCys/tHcy will assist in identifying heterozygotes. Boddie, et al., Metabolism (1998) 47:207-211.
In addition, total cysteine levels are correlated with age, total cholesterol concentration, diastolic blood pressure and coffee consumption, but unlike homocysteine levels, not smoking status, folate and vitamin intake, heart rate and physical activity. El-Khairy, et al., Am. J. Clin. Nutr. (1999) 70:1016-1024. Both tCys and tHcy are associated with renal failure. Mansoor, et al., Clin. Chem. (1993) 39:980-985.
Methods to determine total homocysteine enzymatically have also been described, for example, in U.S. Pat. No. 6,468,762 issued 22 Oct. 2002 as well as U.S. Pat. Nos. 6,066,467; 5,998,191; and 5,985,540, the disclosures of which are incorporated herein by reference.
An alternative method of measuring total cysteine is also described in the above mentioned '762 patent wherein the sample to be assayed is treated with a non-specific desulfurase. To measure cysteine directly, S-adenosyl homocysteine hydrolase (SAHH) and adenosine are added to the sample containing a non-specific desulfurase, where the SAHH is present in sufficient quantity to catalyze the conversion of all of the homocysteine in the sample to SAH, thus protecting it from the action of the desulfurase. Thus, only cysteine levels in the sample are measured.
The present invention provides an alternative enzymatic based assay for total cysteine. The availability of both assays permits not only each total homocysteine and total cysteine to be determined, but also the tCys/tHcy ratio.